1. Field of the Invention
This invention involves the use of novel virulence-specific genes of Listeria monocytogenes as targets for specific diagnosis and potential control of virulent strains of L. monocytogenes. More particularly, this invention provides a PCR or hybridization method, which uses specific primers or probes corresponding to virulence-specific genes for the identification and control of virulent strains of Listeria monocytogenes. 
2. Background of the Technology
L. monocytogenes is an important cause of human food borne diseases world wide. A notable feature of L. monocytogenes is that it shows considerable variation in its ability to produce listeriosis. On the one extreme, some L. monocytogenes strains are virulent and can result in severe disease and mortality. On the other, some have limited capability to establish in the host and are relatively avirulent and harmless. Because manufactured food products detected with L. monocytogenes are recalled or downgraded (i.e., used for pet food), contamination with this species may render significant economic losses. With outbreaks of listeriosis due to contaminated foods on the increase in recent years, L. monocytogenes has become a major concern to the food industry and health regulation authority.
Apart from adapting stringent quality control measures during food processing procedures, frequent monitoring with specific laboratory tests for virulent strains of L. monocytogenes is vital in reducing unnecessary food product recalls and allaying consumer concerns. The current diagnostic methods are incapable of distinguishing virulent from avirulent strains of L. monocytogenes. 
The complete genome of Listeria monocytogenes EGDe strain was reported recently (Glaser et al., 2001). Although this publication contains a list of all known and putative genes in L. monocytogenes EGD strain as well as their nucleotide sequences, it does not provide any information on the actual application of these genes. Therefore, although the DNA sequences of the genes described in this invention have been published and are in public domain through the release of the L. monocytogenes EGDe genome sequence, there are no prior publications on the functions of these genes or on their use for research or diagnostic purposes.
Previous research used PCR and DNA sequencing or restriction fragment length polymorphism of the L. monocytogenes hlyA, actA, and inlA genes to group L. monocytogenes into three genetic lineages, with the various lineages varying in potential for human virulence (Norton et al., 2001; Wiedmann et al., 1997). Ribotyping (sequencing of rRNA genes) was also used in this research. These assays are different from the present assay employed by the inventors in that they require either DNA sequencing or restriction digests following PCR amplification, while the present assay is simply a PCR assay. In addition, the hlyA, actA, and inlA genes are present in all L. monocytogenes isolates, while the virulence-specific genes described by the inventors are found only in virulent strains of L. monocytogenes. 
Another PCR assay, random amplification of polymorphic DNA (RAPD) PCR, has been used to classify L. monocytogenes into genetic groups that tend to predict virulence. This technique is based on the use of nonspecific primers that bind to unknown sequences in the L. monocytogenes chromosome (Franciosa et al., 2001). The PCR assay employed by the inventors is based on primers that bind to specific virulence associated chromosomal sequences that we have identified.
Other assays have been described for differentiation of virulent and avirulent L. monocytogenes isolates. The “gold standard” for virulence testing of L. monocytogenes isolates is the mouse virulence test. This test is expensive, labor intensive, requires several weeks to complete, and requires regulatory approval to ensure humane treatment of animals. Assays have been described based on cell culture models; one correlated L. monocytogenes virulence with the ability of isolates to form plaques on HT-29 cells (Roche et al., 2001), and another correlated virulence with the ability to cause cytopathogenic effects in Caco-2 cells (Pine et al., 1991). Although the use of cell culture models represents an improvement over mouse virulence testing, it is still time-consuming and labor intensive.
Research has been published on the use of phenotypic detection of virulence factor expression (listeriolysin, phosphatidylinositol phospholipase C, phosphatidylcholine phospholipase C) to separate virulent from avirulent L. monocytogenes isolates (Erdenlig et al., 2000). Research has also been published on the use of monoclonal antibodies for detection of virulence factor expression (listeriolysin and phosphatidylcholine phospholipase C) to distinguish virulent and avirulent isolates (Erdenlig et al., 1999). The dot blot hybridization technique described in this invention has also been previously published. For example, this technique was employed to identify virulence and avirulence associated markers of Dichelobacter nodosus—the ovine footrot pathogen (Liu & Yong, 1993). Several PCR assays have been described for species specific detection of L. monocytogenes (examples include Aznar & Alarcon, 2002; Bassler et al., 1995; Blais et al., 1997; Klein & Juneja, 1997; Norton & Batt, 1999; Winters et al., 1999). PCR assays for distinguishing all six Listeria species can be based on the 16S and 23S rRNA genes (Sallen et al., 1996) or the intergenic spacer region of 16S and 23S rRNA genes (Graham et al., 1997), or the iap gene (Bubert, et al., 1999). However, none of these PCR assays distinguish virulent L. monocytogenes isolates from avirulent isolates.